Interaction of cytosol fractions containing activated glucocorticoid-receptor complexes from rat liver and thymus with heterologous nuclei: effects on transcription.

Two rat liver cytosol fractions containing activated glucocorticoid-receptor complexes are able to stimulate the transcriptional activity of rat liver nuclei; the respective fractions from the cytosol of thymocytes inhibit the capacity of thymus nuclei for RNA synthesis. A similar inhibitory effect on thymus nuclei is exerted by the presence of rat liver cytosol fractions. Spot hybridization using a tyrosine aminotransferase (TAT) probe demonstrates that TAT gene expression is stimulated by the liver cytosol fractions acting on homologous nuclei whereas it is inhibited, in thymus nuclei, by the addition of thymus cytosol fractions. No effect on transcription is observed if the liver or thymus cytosol is heat activated in the presence of the glucocorticoid antagonist, cortexolone. Treatment of liver nuclei, previously subjected to the action of thymus cytosol fractions with the respective liver ones, restores transcriptional activity to control or higher levels. We conclude that rat thymocyte nuclei and cytosol contain transcriptional factors, which in the presence of the glucocorticoid-receptor complex, irrespective of its source, inhibit gene expression, whereas in the absence of such factors, the glucocorticoid-receptor complex positively regulates the respective genes.

Insulin enhances glucocorticoid induction of gene expression in a sequence specific manner.

Glucocorticoid receptor (GR) induces transcriptional activation of specific genes by binding to cis-acting enhancer sequences termed glucocorticoid responsive elements (GREs). Insulin regulates gene transcription part of which is mediated sequences dominated by GREs. By transient expression in HTC cells of fusion gene consist MMTV 5' flanking sequences containing the GREs on the thymidine kinase promoter (TK) and the coding region of the chloramphenicol acetyl transferase gene (CAT), we demonstrate that insulin increases 2-fold the produced induction from dexamethasone, while insulin alone does not exceed any significant effect. This synergistic effect of insulin is sequence specific since, expression of the glucocorticoid unresponsive gene RSV-CAT at the same cell line is affected neither by insulin nor dexamethasone or both. Transient expression of CAT constructs with progressive 5' deletion mutants of mouse mammary tumor virus (MMTV) promoter, fail to detect a cis-acting insulin responsive DNA element, that participates in increased dexamethasone mediated induction. We believe that insulin activates an intracellular mediator-transcription factor, that stimulates gene expression indirectly, by interaction with the glucocorticoid receptor.

The effect of partially purified dexamethasone receptor on RNA synthesis in rat thymus and liver nuclei.

DEAE Sephadex-A-50 chromatography of rat liver and thymus cytosol charged with dexamethasone (0.1 microM) and heated at 25 degrees C for 30 min, separates two main hormone--receptor complexes, one in the flow through (DE-1) and one eluting with 150 mM [Cl-] (DE-2). Incubation of these two fractions with homologous nuclei results in a 20% stimulation and a 20-25% inhibition of transcription of liver and thymus nuclei, respectively. No difference was observed between the action of DE-1 and DE-2. If the DE-1 and DE-2 fractions of cytosol charged with the glucocorticoid antagonist, cortexolone, are used under the same experimental conditions, no effect on the transcriptional activity of liver or thymus nuclei can be observed. The fact that from nuclei only DE-1 can be isolated, not DE-2, suggests that DE-1 is the intranuclearly active form and that DE-2, with a still unknown mechanism, is transformed to DE-1. A linear relation exists between the number of GR-complexes (DE-1 or DE-2) translocated and the change in RNA synthesis. Transcriptional effects start when a critical number of acceptor sites are occupied in the nucleus (approximately 200-300 sites/nucleus) for both receptor forms.